Transition metal ions function as required cofactors for myriad metalloenzymes that catalyze a large diversity of biological reactions. These same metal ions, however, are cytotoxic in excess. As a result, all cells dedicate considerable regulatory machinery to maintaining the intracellular concentrations and types of metal ions, i.e., homeostasis, in order to quickly adapt to a changing microenvironment. Emerging evidence suggests that microbial metal ion homeostasis and resistance to toxicity defines a critical component in human host-bacterial pathogen interactions during infection. This proposal seeks continuing support for a project whose long-term goal is to understand the molecular mechanisms of this adaptive response, including how metal ions are sensed by metalloregulatory or "metal sensor" proteins, and trafficked in the cell, and how perturbations in metal metabolism impact bacterial pathogenesis and virulence. For the next project obacterium tuberculosis and Streptococcus pneumoniae. Our specific aims are to: 1) Elucidate the structural and thermodynamic basis of Zn-regulation by S. aureus CzrA, a model ArsR-family repressor of the czr (chromosomal zinc-regulated) operon; 2) Employ protein ligation strategies to site-specifically incorporate amino acid analogs to directly test our allosteric coupling model for CzrA; 3) Use a panel of CzrA mutants to critically evaluate the functional relationship between KZn, the Zn binding affinity of a metal sensor, and the ability to sense zinc in the cell; 4) Elucidate the structural determinants of DNA binding and Cu-mediated allosteric regulation of DNA binding by the novel Cu-sensor M. tuberculosis CsoR, the repressor of the Cu-effluxing copper-sensitive operon (cso); and 5) Structurally characterize S. pneumoniae AdcR, a postulated novel Zn-sensor that regulates the expression of a zinc uptake system (adc operon) required for adhesion competence and virulence in pneumohost-bacterial pathogen interactions during[unreadable] infection. The research outlined in this proposal seeks continuing support of a project designed to understand the structure and mechanism of the intracellular regulators of this process, termed "metal sensor" or metalloregulatory proteins, that control the expression of genes linked to this adaptive response. Completion of these studies may ultimately lead to novel strategies for the targeted development of antimicrobial agents that exploit or induce misregulation of metal homeostasis. 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